The long-term goal of this application is to understand the antigen processing mechanism by which endogenously synthesized proteins yield precisely cleaved peptide/MHC class I complexes on the cell surface. The expression of these peptide/MHC I complexes is essential for development and maintenance of the CD8 cytotoxic T-cell repertoire and for immune responses specific for tumors, intracellular pathogens, allogeneic tissue grafts and self-tissues in autoimmunity. Failure to express peptide/MHC complexes is an important mechanism by which tumor cells and pathogens escape immune surveillance. We have developed novel cellular, biochemical and genetic techniques for analysis of naturally processed antigenic peptides. These techniques allow the direct analysis of the final processed peptides products, and for the first time, their proteolytic intermediates that are generated in the cytosol and in the ER. Here we propose to test different hypotheses on, (a) how the antigen processing steps in the cytosol and in the ER shape this pool of naturally processed peptides available for presentation by MHC class I molecules, and (b) how peptide trimming in the ER results in the generation and display of precisely cleaved peptides by the MHC I molecules on the cell surface. We anticipate that the answer to these questions will improve our understanding of the key factors that determine epitope selection, epitope dominance and the efficiency of the antigen processing pathway.